A variety of mutagenesis techniques in the mouse are available to understand gene function and to develop animal models of human disease. However, the mouse genetics community has lacked a broadly applicable means of performing phenotypic screens to identify mutations in specific biochemical or genetic pathways. The development of cell-based phenotypic screens in mouse embryonic stem (ES) cells could provide the critical methodology for these types of focused mutation screens. The TGF-beta-related signaling pathways would be excellent candidates for directed phenotypic screens in ES cells. These signaling pathways play a critical role in a variety of disease states, including tumor progression. The diverse roles that these signaling systems play in both embryonic development and disease suggests a complex mode of regulation. The nature of how these signals are regulated in an in vivo context is still largely unknown. A directed mutation screen of TGF-beta-related signaling processes would generate valuable animal models to identify and further understand the regulators of these pathways. Many of the components that mediate TGF-beta signaling are present on mouse chromosome 18, suggesting the possibility of a chromosomal-directed phenotypic screen for TGF-beta signaling mutations. In this proposal, reagents will be developed to test the hypothesis that a mutation in mouse ES cells can be identified based on the altered activity of the mutant gene product, and that this altered activity can be assayed using a downstream reporter. A screening strategy will be developed for identifying ES cells that carry chemically induced mutations in the immediate early response components of TGF-beta superfamily signaling on mouse chromosome 18. A luciferase based reporter will be used as a readout of the responsiveness of ES cells to TGF-beta signaling. Mutagenesis will be accomplished with ethylnitrosourea, a highly effective mutagen in ES cells. To resolve recessive mutations, a Cre-loxP-mediated mitotic recombination system will be utilized to generate cells that are homozygous for a mutagenized chromosome 18. Cell-based mutation screening strategies as developed in this proposal can be applied to any active biochemical pathway in mouse ES cells, to generate mouse lines for analysis in vivo. [unreadable] [unreadable] [unreadable]